The major objective of this project is to compare the structural and regulatory characteristics of the mitochondrial and cytosolic varieties of P-enolpyruvate carboxy-kinase (PEPCK) to determine whether the two forms may have different genetic and metabolic controls. Up until the proposed studies, little knowledge is available concerning the comparative structure of the two forms from the same tissue. We have isolated both types of the enzyme in pure form from Rhesus monkey liver and prepared antibodies against both antigens. The results indicate that the two forms are similar in size but can be separated electrophoretically and do not cross-react immunologically. The plans for the coming year are to compare the structures of the two isozymes by amino acid analysis, end-group determinations and peptide mapping to establish the degree of homology. The possibility that the catalytic sites of the enzyme may be similar will be studied, initally by kinetic mapping of the PEP site through the use of a series of PEP analogs which have been demonstrated to be alternative substrates or inhibitors. The regulation of the mitochondrial isozyme will be studied in guinea pigs by comparing the rates of synthesis and degradation of the two isozymes in fed, fasted and hormone-stimulted animals.